Comparative transcriptome and metabolome analysis reveal key regulatory defense networks and genes involved in enhanced salt tolerance of Actinidia (kiwifruit)

Abstract

The Actinidia (kiwifruit) is an emerging fruit plant that is severely affected by salt stress in northern China. Plants have evolved several signaling network mechanisms to cope with the detrimental effects of salt stress. To date, no reported work is available on metabolic and molecular mechanisms involved in kiwifruit salt tolerance. Therefore, the present study aims to decipher intricate adaptive responses of two contrasting salt tolerance kiwifruit species Actinidia valvata [ZMH (an important genotype), hereafter referred to as R] and Actinidia deliciosa [‘Hayward’ (an important green-fleshed cultivar), hereafter referred to as H] under 0.4% (w/w) salt stress for time courses of 0, 12, 24, and 72 hours (hereafter refered to as h) by combined transcriptome and metabolome analysis. Data revealed that kiwifruit displayed specific enrichment of differentially expressed genes (DEGs) under salt stress. Interestingly, roots of R plants showed a differential expression pattern for up-regulated genes. The KEGG pathway analysis revealed the enrichment of DEGs related to plant hormone signal transduction, glycine metabolism, serine and threonine metabolism, glutathione metabolism, and pyruvate metabolism in the roots of R under salt stress. The WGCNA resulted in the identification of five candidate genes related to glycine betaine (GB), pyruvate, total soluble sugars (TSS), and glutathione biosynthesis in kiwifruit. An integrated study of transcriptome and metabolome identified several genes encoding metabolites involved in pyruvate metabolism. Furthermore, several genes encoding transcription factors were mainly induced in R under salt stress. Functional validation results for overexpression of a candidate gene betaine aldehyde dehydrogenase (AvBADH, R_transcript_80484 ) from R showed significantly improved salt tolerance in Arabidopsis thaliana (hereafter referred to as At) and Actinidia chinensis [‘Hongyang’ (an important red-fleshed cultivar), hereafter referred to as Ac] transgenic plants than in WT plants. All in all, salt stress tolerance in kiwifruit roots is an intricate regulatory mechanism that consists of several genes encoding specific metabolites.

References

[1] Samsatly J, Copley TR, Jabaji SH . Antioxidant genes of plants and fungal pathogens are distinctly regulated during disease development in different Rhizoctonia solani pathosystems. PLoS One. 2018; 13: e0192682.

[2] Zhao C, Jiang W, Zayed O et al. The LRXs-RALFs-FER module controls plant growth and salt stress responses by modulating multiple plant hormones . Natl Sci Rev. 2021; 8: nwaa149.

[3] Zhao R, Sun H, Mei C et al. The Arabidopsis Ca²⁺-dependent protein kinase CPK12 negatively regulates abscisic acid signaling in seed germination and post-germination growth. New Phytol. 2011; 192: 61-73.

[4] Mackay A. Climate change 2007: impacts, adaptation and vulnerability. Contribution of working group II to the fourth assessment report of the intergovernmental panel on climate change. J Environ Qual. 2007; 37: 2407.

[5] Golldack D, Li C, Mohan H et al. Tolerance to drought and salt stress in plants: unraveling the signaling networks. Front Plant Sci. 2014; 5: 151.

[6] Agarwal PK, Shukla PS, Gupta K et al. Bioengineering for salinity tolerance in plants: state of the art. Mol Biotechnol. 2013; 54: 102-23.

[7] Miller GAD, Suzuki N, Ciftci-Yilmaz S et al. Reactive oxygen species homeostasis and signalling during drought and salinity stresses. Plant Cell Environ. 2010; 33: 453-67.

[8] Li HW, Zang BS, Deng XW et al. Overexpression of the trehalose-6-phosphate synthase gene OsTPS1 enhances abiotic stress tolerance in rice . Planta. 2011; 234: 1007-18.

[9] Cotsaftis O, Plett D, Johnson AAT et al. Root-specific transcript profiling of contrasting rice genotypes in response to salinity stress. Mol Plant. 2011; 4: 25-41.

[10] Kumar K, Kumar M, Kim SR et al. Insights into genomics of salt stress response in rice. Rice. 2013; 6: 27.

[11] Kumar S, Dhingra A, Daniell H . Plastid-expressed betaine aldehyde dehydrogenase gene in carrot cultured cells, roots, and leaves confers enhanced salt tolerance . Plant Physiol. 2004; 136: 2843-54.

[12] Qiao WH, Zhao XY, Li W et al. Overexpression of AeNHX1, a root-specific vacuolar Na⁺/H⁺ antiporter from Agropyron elongatum, confers salt tolerance to Arabidopsis and Festuca plants. Plant Cell Rep. 2007; 26: 1663-72.

[13] Saad RB, Zouari N, Ben Ramdhan W et al. Improved drought and salt stress tolerance in transgenic tobacco overexpressing a novel A20/AN1 zinc-finger “ AlSAP” gene isolated from the halophyte grass Aeluropus littoralis . Plant Mol Biol. 2010; 72: 171-90.

[14] Hmida-Sayari A, Gargouri-Bouzid R, Bidani A et al. Overexpression of Δ1-pyrroline-5-carboxylate synthetase increases proline production and confers salt tolerance in transgenic potato plants . Plant Sci. 2005; 169: 746-52.

[15] Jing P, Zou J, Kong L et al. OsCCD1, a novel small calcium-binding protein with one EF-hand motif, positively regulates osmotic and salt tolerance in rice . Plant Sci. 2016; 247: 104-14.

[16] Gao Y, Liu J, Yang F et al. The WRKY transcription factor WRKY8 promotes resistance to pathogen infection and mediates drought and salt stress tolerance in Solanum lycopersicum . Physiol Plant. 2020; 168: 98-117.

[17] Li M, Chen R, Jiang Q et al. GmNAC06, a NAC domain transcription factor enhances salt stress tolerance in soybean . Plant Mol Biol. 2020; 105: 333-45.

[18] Han D, Han J, Yang G et al. An ERF transcription factor gene from Malus baccata (L.) borkh, MbERF11, affects cold and salt stress tolerance in Arabidopsis . Forests. 2020; 11: 514.

[19] Wu J, Yu C, Hunag L et al. Overexpression of MADS-box transcription factor OsMADS25 enhances salt stress tolerance in Rice and Arabidopsis . Plant Growth Regul. 2020; 90: 163-71.

[20] Shulaev V, Cortes D, Miller G et al. Metabolomics for plant stress response. Physiol Plant. 2008; 132: 199-208.

[21] Gupta B, Huang B . Mechanism of salinity tolerance in plants: physiological, biochemical, and molecular characterization. Int J Genomics. 2014; 2014: 1-18.

[22] Patel MK, Kumar M, Li W et al. Enhancing salt tolerance of plants: from metabolic reprogramming to exogenous chemical treatments and molecular approaches. Cell. 2020; 9: 2492.

[23] Pan J, Li Z, Dai S et al. Integrative analyses of transcriptomics and metabolomics upon seed germination of foxtail millet in response to salinity. Sci Rep. 2020; 10: 1-16.

[24] Meng X, Liu S, Dong T et al. Comparative transcriptome and proteome analysis of salt-tolerant and salt-sensitive sweet potato and overexpression of IbNAC7 confers salt tolerance in Arabidopsis . Front Plant Sci. 2020; 11: 1342.

[25] Xing JC, Zhao BQ, Dong J et al. Transcriptome and metabolome profiles revealed molecular mechanisms underlying tolerance of Portulaca oleracea to saline stress . Russ J Plant Physiol. 2020; 67: 146-52.

[26] Wei T, Wang Y, Liu JH . Comparative transcriptome analysis reveals synergistic and disparate defense pathways in the leaves and roots of trifoliate orange (Poncirus trifoliata) autotetraploids with enhanced salt tolerance . Hortic Res. 2020; 7: 1-14.

[27] Huang S, Ding J, Deng D et al. Draft genome of the kiwifruit Actinidia chinensis . Nat Commun. 2013; 4: 1-9.

[28] Wang T, Gleave AP . Applications of biotechnology in kiwifruit (Actinidia) . In: Agbo EC, ed. Innovations in Biotechnology.InTech, 2012, 3-30.

[29] Abid M, Zhang YJ, Li Z et al. Effect of salt stress on growth, physiological and biochemical characters of four kiwifruit genotypes. Sci Hortic. 2020; 271: 109473.

[30] Zhong YP, Xiu-Juan QI, Chen JY et al. Growth and physiological responses of four kiwifruit genotypes to salt stress and resistance evaluation. J Integr Agric. 2019; 18: 83-95.

[31] Cimini S, Locato V, Giacinti V et al. A multifactorial regulation of glutathione metabolism behind salt tolerance in rice. Antioxidants. 2022; 11: 1114.

[32] Yan H, Nie Y, Cui K et al. Integrative transcriptome and metabolome profiles reveal common and unique pathways involved in seed initial imbibition under artificial and natural salt stresses during germination of Halophyte quinoa . Front Plant Sci. 2022; 13: 853326.

[33] Nahar K, Hasanuzzaman M, Alam MM et al. Roles of exogenous glutathione in antioxidant defense system and methylglyoxal detoxification during salt stress in mung bean. Biol Plant. 2015; 59: 745-56.

[34] Zhao X, Chen Z, Leng P et al. iTRAQ-based quantitative proteomic analysis of the response of Hylotelephium erythrostictum leaves to salt stress . Sci Hortic. 2020; 264: 109190.

[35] Liu D, Hu R, Zhang J et al. Overexpression of an agave phosphoenolpyruvate carboxylase improves plant growth and stress tolerance . Cell. 2021; 10: 582.

[36] De la Torre-González A, Montesinos-pereira D, Blasco B et al. Influence of the proline metabolism and glycine betaine on tolerance to salt stress in tomato (Solanum lycopersicum L.) commercial genotypes . J Plant Physiol. 2018; 231: 329-36.

[37] Rady MOA, Semida WM, Abd El-Mageed TA et al. Up-regulation of antioxidative defense systems by glycine betaine foliar application in onion plants confer tolerance to salinity stress. Sci Hortic. 2018; 240: 614-22.

[38] Missihoun TD, Willée E, Guegan JP et al. Overexpression of ALDH10A8 and ALDH10A9 genes provides insight into their role in glycine betaine synthesis and affects primary metabolism in Arabidopsis thaliana . Plant Cell Physiol. 2015; 56: 1798-807.

[39] Wang WR, Liang JH, Wang GF et al. Overexpression of PpSnRK1α in tomato enhanced salt tolerance by regulating ABA signaling pathway and reactive oxygen metabolism . BMC Plant Biol. 2020; 20: 1-15.

[40] Zhang J, Yu H, Zhang Y et al. Increased abscisic acid levels in transgenic maize overexpressing AtLOS5 mediated root ion fluxes and leaf water status under salt stress . J Exp Bot. 2016; 67: 1339-55.

[41] Fang Q, Jiang T, Xu L et al. A salt-stress-regulator from the poplar R2R3 MYB family integrates the regulation of lateral root emergence and ABA signaling to mediate salt stress tolerance in Arabidopsis . Plant Physiol Biochem. 2017; 114: 100-10.

[42] Wang T, Tohge T, Ivakov A et al. Salt-related MYB1 coordinates abscisic acid biosynthesis and signaling during salt stress in Arabidopsis . Plant Physiol. 2015; 169: 1027-41.

[43] Li Z, Zhong Y, Bai D et al. Comparative analysis of physiological traits of three Actinidia valvata Dunn. Genotypes during waterlogging and post-waterlogging recovery . Hortic Environ Biotechnol. 2020; 61: 825-36.

[44] Raghavendra AS, Gonugunta VK, Christmann A et al. ABA perception and signalling. Trends Plant Sci. 2010; 15: 395-401.

[45] Javid MG, Sorooshzadeh A, Moradi F et al. The role of phytohormones in alleviating salt stress in crop plants. Aust J Crop Sci. 2011; 5: 726-34.

[46] Ming R, Zhang Y, Wang Y et al. The JA-responsive MYC2-BADH-like transcriptional regulatory module in Poncirus trifoliata contributes to cold tolerance by modulation of glycine betaine biosynthesis. New Phytol. 2020; 229: 2730-50.

[47] Sharma I, Ching E, Saini S et al. Exogenous application of brassinosteroid offers tolerance to salinity by altering stress responses in rice variety ’Pusa Basmati-1′. Plant Physiol Biochem. 2013; 69: 17-26.

[48] Zhu T, Deng X, Zhou X et al. Ethylene and hydrogen peroxide are involved in brassinosteroid-induced salt tolerance in tomato. Sci Rep. 2016; 6: 1-15.

[49] Zhao S, Zhang Q, Liu M et al. Regulation of plant responses to salt stress. Int J Mol Sci. 2021; 22: 4609.

[50] Verma V, Ravindran P, Kumar PP . Plant hormone-mediated regulation of stress responses. BMC Plant Biol. 2016; 16: 1-10.

[51] Zhao Y, Zhang Z, Gao J et al. Arabidopsis duodecuple mutant of PYL ABA receptors reveals PYL repression of ABA-independent SnRK2 activity . Cell Rep. 2018; 23: 3340-3351.e5.

[52] Barrero JM, Rodríguez PL, Quesada V et al. Both abscisic acid (ABA)-dependent and ABA-independent pathways govern the induction of NCED3, AAO3 and ABA1 in response to salt stress . Plant Cell Environ. 2006; 29: 2000-8.

[53] Hamayun M, Khan SA, Khan AL et al. Effect of salt stress on growth attributes and endogenous growth hormones of soybean cultivar ’Hwangkeumkong’. Pakistan J Bot. 2010; 42: 3103-12.

[54] Ashraf M, Foolad MR . Roles of glycine betaine and proline in improving plant abiotic stress resistance. Environ Exp Bot. 2007; 59: 206-16.

[55] Rosa M, Prado C, Podazza G et al. Soluble sugars: metabolism, sensing, and abiotic stress. Plant Signal Behav. 2009; 4: 388-93.

[56] Sami F, Yusuf M, Faizan M et al. Role of sugars under abiotic stress. Plant Physiol Biochem. 2016; 109: 54-61.

[57] Wei D, Zhang W, Wang C et al. Genetic engineering of the biosynthesis of glycinebetaine leads to alleviate salt-induced potassium efflux and enhances salt tolerance in tomato plants. Plant Sci. 2017; 257: 74-83.

[58] Li D, Zhang T, Wang M et al. Genetic engineering of the biosynthesis of glycine betaine modulates phosphate homeostasis by regulating phosphate acquisition in tomato. Front Plant Sci. 2019; 9: 1995.

[59] Chen THH, Murata N . Glycinebetaine protects plants against abiotic stress: mechanisms and biotechnological applications. Plant Cell Environ. 2011; 34: 1-20.

[60] Niazian M, Sadat-Noori SA, Tohidfar M et al. Betaine aldehyde dehydrogenase (BADH) vs. Flavodoxin (Fld): two important genes for enhancing plants stress tolerance and productivity . Front Plant Sci. 2021; 12: 650215.

[61] Naliwajski MR, Skłodowska M . The oxidative stress and antioxidant systems in cucumber cells during acclimation to salinity. Biol Plant. 2014; 58: 47-54.

[62] Hasanuzzaman M, Nahar K, Anee TI et al. Glutathione in plants: biosynthesis and physiological role in environmental stress tolerance. Physiol Mol Biol Plants. 2017; 23: 249-68.

[63] Shi H, Ye T, Chan Z . Comparative proteomic responses of two bermudagrass (Cynodon dactylon (L). Pers.) varieties contrasting in drought stress resistance . Plant Physiol Biochem. 2014; 82: 218-28.

[64] Sobhanian H, Motamed N, Jazii FR et al. Salt stress induced differential proteome and metabolome response in the shoots of Aeluropus lagopoides (Poaceae), a halophyte C4 plant. J Proteome Res. 2010; 9: 2882-97.

[65] Murashige T, Skoog F . A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant. 1962; 15: 473-97.

[66] He L, Jing Y, Shen J et al. Mitochondrial pyruvate carriers prevent cadmium toxicity by sustaining the TCA cycle and glutathione synthesis. Plant Physiol. 2019; 180: 198-211.

[67] Liu N, Ding Y, Fromm M et al. Endogenous ABA extraction and measurement from Arabidopsis leaves . Bio Protoc. 2014; 4: e1257.

[68] Moreno-Galván AE, Cortés-Patiño S, Romero-Perdomo F et al. Proline accumulation and glutathione reductase activity induced by drought-tolerant rhizobacteria as potential mechanisms to alleviate drought stress in Guinea grass . Appl Soil Ecol. 2020; 147: 103367.

[69] Kim D, Paggi JM, Park C et al. Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat Biotechnol. 2019; 37: 907-15.

[70] Zheng Y, Jiao C, Sun H et al. iTAK: a program for genome-wide prediction and classification of plant transcription factors, transcriptional regulators, and protein kinases. Mol Plant. 2016; 9: 1667-70.

[71] Love M, Huber W, Anders S . Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014; 15: 550.

[72] Robinson MD, McCarthy DJ, Smyth GK . edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics. 2010; 7: 139-40.

[73] Young MD, Wakefield MJ, Smyth GK et al. Gene ontology analysis for RNA-seq: accounting for selection bias. Genome Biol. 2010; 11: R14.

[74] Mao X, Tao C, Olyarchuk JG et al. Automated genome annotation and pathway identification using the KEGG Orthology (KO) as a controlled vocabulary. Bioinformatics. 2005; 21: 3787-93.

[75] Zhang Y, Wang P, Xia H et al. Comparative transcriptome analysis of basal and zygote-located tip regions of peanut ovaries provides insight into the mechanism of light regulation in peanut embryo and pod development. BMC Genomics. 2016; 17: 606.

[76] Li Y, Fang J, Qi X et al. Combined analysis of the fruit metabolome and transcriptome reveals candidate genes involved in flavonoid biosynthesis in Actinidia arguta . Int J Mol Sci. 2018; 19: 1471.

[77] Livak KJ, Schmittgen TD . Analysis of relative gene expression data using real-time quantitative PCR and the 2^−ΔΔCT method. Methods. 2001; 25: 402-8.

[78] Langfelder P, Horvath S . WGCNA: an R package for weighted correlation network analysis. BMC Bioinformatics. 2008; 9: 1-13.

[79] Langfelder P, Zhang B, Horvath S . Defining clusters from a hierarchical cluster tree: the dynamic tree cut package for R. Bioinformatics. 2008; 24: 719-20.

[80] Shannon P, Markiel A, Ozier O et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 2003; 13: 2498-504.

[81] Giavalisco P, Köhl K, Hummel J et al. 13C isotope-labeled metabolomes allowing for improved compound annotation and relative quantification in liquid chromatography-mass spectrometry-based metabolomic research. Anal Chem. 2009; 81: 6546-51.

[82] Cuadros-Inostroza A, Lisec J, Redestig H, Hannah MA . The TargetSearch Package. Viewpoints 2013.

[83] Kassambara A, Mundt F . Factoextra: Extract and Visualize the Results of Multivariate Data Analyses. 2020. (Retrieved from https://github.com/kassambara/factoextra).

[84] Chong J, Wishart DS, Xia J . Using MetaboAnalyst 4.0 for comprehensive and integrative metabolomics data analysis. Curr Protoc Bioinforma. 2019; 68: e86.

[85] Junker BH, Klukas C, Schreiber F . VANTED: a system for advanced data analysis and visualization in the context of biological networks. BMC Bioinformatics. 2006; 7: 1-13.

[86] Davis AM, Hall A, Millar AJ et al. Protocol: streamlined subprotocols for floral-dip transformation and selection of transformants in Arabidopsis thaliana . Plant Methods. 2009; 5: 3-7.

[87] Wang T, Atkinson R, Janssen B . Choice of agrobacterium strain for transformation of kiwifruit . Acta Hortic. 2007; 753: 227-32.

[88] Li X, Pan Y, Chang B et al. NO promotes seed germination and seedling growth under high salt may depend on EIN3 protein in Arabidopsis . Front Plant Sci. 2016; 6: 1203.

[89] Ganguly M, Datta K, Roychoudhury A et al. Overexpression of Rab16A gene in indica rice variety for generating enhanced salt tolerance . Plant Signal Behav. 2012; 7: 502-9.

[90] Zhang M, Fang Y, Ji Y et al. Effects of salt stress on ion content, antioxidant enzymes and protein profile in different tissues of Broussonetia papyrifera . South African J Bot. 2013; 85: 1-9.

[91] Nazar R, Iqbal N, Syeed S et al. Salicylic acid alleviates decreases in photosynthesis under salt stress by enhancing nitrogen and sulfur assimilation and antioxidant metabolism differentially in two mungbean cultivars. J Plant Physiol. 2011; 168: 807-15.

[92] Ali MB, Hahn EJ, Paek KY . Effects of light intensities on antioxidant enzymes and malondialdehyde content during short-term acclimatization on micropropagated Phalaenopsis plantlet . Environ Exp Bot. 2005; 54: 109-20.

[93] Ji W, Zhu Y, Li Y et al. Over-expression of a glutathione S-transferase gene, GsGST, from wild soybean (Glycine soja) enhances drought and salt tolerance in transgenic tobacco . Biotechnol Lett. 2010; 32: 1173-9.

[94] Chen K, Chen L, Fan J et al. Alleviation of heat damage to photosystem II by nitric oxide in tall fescue. Photosynth Res. 2013; 116: 21-31.

[95] Ja’afar U, Aliero AA, Yahaya TO . A study on proline and glycine betaine contents as salinity and drought tolerant indicators in Solanum lycopersicum L. (cultivar: ’Roma’) . Glob J Sci Front Res D Agric Vet. 2018; 18: 29-32.

[96] Huang XS, Wang W, Zhang Q et al. A basic helix-loop-helix transcription factor PtrbHLH of Poncirus trifoliata confers cold tolerance and modulates POD-mediated scavenging of H₂O₂ . Plant Physiol. 2013; 162: 1178-94.

[97] Riechmann J, Heard J, Martin G et al. Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes . Science. 2000; 290: 2105-10.

[98] Mukherjee SP, Choudhuri MA . Implications of water stress-induced changes in the levels of endogenous ascorbic acid and hydrogen peroxide in Vigna seedlings . Physiol Plant. 1983; 58: 166-70.

[99] Hernandez JA, Ferrer MA, Jimenez A et al. Antioxidant systems and O₂^•−/H₂O₂ production in the apoplast of pea leaves. Its relation with salt-induced necrotic lesions in minor veins . Plant Physiol. 2001; 127: 817-31.

[100] Qu Y, Nong Q, Jian S et al. An AP2/ERF gene, HuERF1, from pitaya (Hylocereus undatus) positively regulates salt tolerance . Int J Mol Sci. 2020; 21: 4586.